Molecular motors that drive cargo transport along cytoskeletal filaments are critical for processes such as cell division, cell motility, intracellulr trafficking, subcellular organization, and ciliary function. Defects in motor- driven transport processes are known to contribute to neurodegenerative diseases, cancer, and ciliopathies. A key property of motors that drive cargo transport is the ability to undergo processive motility (th ability to take many steps along the filament before detaching). Yet despite decades of research into kinesin motility, we still have a very limited understanding of how kinesin enzymes convert the energy of ATP hydrolysis into processive motility. Our Preliminary Data demonstrate that we have in hand, for the first time, three kinesin families with high sequence similarity but drasticaly different processivity outputs. Thus, we are uniquely positioned to solve an outstanding structure-function question in the kinesin field. We will determine sequence features of the core kinesin motor domain that are required for processive motion. We will investigate how family-specific substitutions and disease-associated mutations in the core motor domain alter the processivity output. We will examine the fundamental role of processive motion in cargo transport in cells. This work will advance our knowledge of fundamental transport processes in cells and will have broad implications for understanding how molecular variation leads to functional variation within large protein families.